The quantitative determination of acid or alkaline phosphatase and isoenzymes thereof in biological fluids, and particularly in human blood serum, has become very important in the diagnosis and treatment of various physical disorders. Acid phosphatase (ACP) refers to a group of phosphatases with optimal activity below pH 7.0 that catalyze the hydrolysis of many orthophosphoric monoesters. ACP is present in most tissues with the prostate gland having the highest concentration. Increased serum levels of ACP can indicate a number of disease states especially prostate cancer.
Alkaline phosphatase (ALP) refers to a group of nonspecific phosphatases that exhibit optimal activity at alkaline pH. Determination of alkaline phosphatase is very important in the detection of bone and liver diseases. For example, elevated alkaline phosphatase concentrations are often associated with Paget's disease, osteosarcomas, osteomalacia, obstructive jaundice, hepatitis and the like. An early and rapid detection of elevated phosphatase concentrations can then lead to rapid treatment of the causative conditions. As a result, various assay procedures have been developed over the years to provide a quantitative determination of acid or alkaline phosphatase.
A number of analytical procedures have been developed whereby a substrate is hydrolyzed by an enzyme of interest to relase a detectable moiety. These procedures use both colorimetric and fluorometric methods. Fluorometric assays are preferred because of generally greater sensitivity. However, known fluorometric assays are deficient in a number of ways. For example, E.P. Application No. 122,148 (published Oct. 17, 1984) describes an assay for microorganisms using certain coumarin derivatives as substrates which release fluorescent dyes when the substrate is hydrolyzed. The coumarin dyes can be attached to a sugar radical, alcohol radical or a phosphate radical. However, the dyes of this reference have absorption and emission spectra in regions subject to spectral interference from hemoglobin and bilirubin.
A preferred substrate for alkaline phosphatase is p-nitrophenylphosphate which, upon hydrolysis at pH 9.8-10.5, releases p-nitrophenol, which has a maximum absorption at about 400 nm. A number of serum components also absorb in that region of the spectrum.
A preferred substrate for acid phosphatase is thymolphthalein monophosphate which, upon hydrolysis at pH 5.4, releases thymolphthalein which has maximum absorption at about 595 nm. However, alkali must be added to the assay to raise the pH to alkaline values prior to measuring the spectrophotometric signal.
Recently, an acid phosphatase assay was described using improved umbelliferone derivatives (Koller et al, Anal. Biochem., 143, pp. 146-151, 1984). However, the dyes described therein absorb at wavelengths at about 500 nm or below, i.e. in the region of absorption of serum components bilirubin and hemoglobin.
It would be desirable to have substrates which are not subject to the problems of known assays.